MOLECULAR ANALYSIS OF PLATELET INTEGRIN ACTIVATION

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In the course of previous research activities undertaken in laboratories investigating the molecular biology of megakaryocyte development and platelet function, the applicant has developed an interest in pursuing advanced molecular studies of platelet activation. The objective of the proposed research is to identify, clone, and characterize cDNAs encoding effector molecules that participate in the modulation of integrin function in activated platelets. Activation of blood platelets is prerequisite for their full participation in hemostasis and thrombosis (Kroll and Schafer, 1989), and is therefore a hallmark of differentiated function in the megakaryocytic lineage. The applicant seeks to develop a new system for the molecular analysis of signaling mechanisms that mediate one of the cardinal manifestations of platelet activation; agonist-stimulated induction of the fibrinogen (Fg) receptor function of integrin alphaIIbB3 (platelet glycoprotein IIb-IIIa). This phenomenon is dependent upon intracellular signal transduction involving the activity of protein kinase C (PKC) and other signaling molecules, but details of these processes, including the identity of specific intermediary and effector entities that regulate the affinity state of alphaIIbbeta3, are unknown. Although receptor-ligand interactions made possible by alpha IIbbeta3 activation have been studied in detail with biochemical, biophysical, and immunologic methodology, the fundamental mechanistic problem is unsolved. To address this problem, a novel screening strategy has been developed in consultation with Professor Brian Seed (Department of Molecular Biology, Massachusetts General Hospital and harvard Medical School), who is generally acknowledged as a pioneer in the development of this technique (panning), and who will serve as consultant for this phase of the project. A human platelet cDNA library will be transiently expressed in megakaryoblastic human erythroleukemia (HEL) cells, and immunologically screened with alph11bbetta3 ligands that report on the activation state of this integrin. This approach obviates the need for stable expression and thereby circumvents the potential problem of propagating differentiated cells. Another important feature of this strategy is that it permits identification of effector molecules that do not necessarily interact directly with alpha11bbeta3. Preliminary studies of stable HEL cell transfectants have demonstrated that heterologous expression of PKC alpha (which is absent in native HEL cells but abundant in platelets and therefore in terminally differentiated megakaryocytes) results in enhancement of alpha IIbbeta3 function in a cell adhesion assay, demonstrating the feasibility of employing this cell line (HELalpha) in correlative studies of intracellular signaling and integrin affinity modulation. The methodologies required for implementation of the specific aims include: (i) construction, and expression in HEL cells, of a human platelet cDNA library; (ii) identification, and isolation from this library, of candidate cDNAs by immunologic screening and expression cloning; and (iii) molecular characterization of candidate cDNAs by nucleotide sequence analysis and expression in megakaryoblastic and other nucleated cell lines that express alphaiibbeta3. The research environment in which the proposed project is to be undertaken consists of a laboratory that is active in the characterization of the molecular mechanisms underlying intracellular signaling processes in platelets and cells of megakaryoblastic lineage and is an integral part of the Vascular Medicine univ founded by the preceptor at Beth Israel Hospital.